Nystatin and method of producing it



June 25, 1957 E. L. HAZEN EI'AL 7 2,

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CENI7I'F"IFUGE P SU E INVENTORS ELIZABETH L. HAZEN ERACHEL F. BROWNPRECIPITATE DRY WITH ETHER BY 2 f NYSTATIN ATTORNEYS June 25, 1957 i E.L. HAZEN EI'AL 2,797,133

' NYS'IATVIN AND METHOD OF PRODUCING IT Fi le dJan. 7.1952 v 2Sheets-Sheet 2 INFRA RED ABSORPTION SPECTRUM INVENTORS ELIZABETH L.HAZEN RACHEL F. BROWN ATTORNEY 2,797,183 NYSTATIN AND METHOD OFPRODUCING IT Elizabeth L. Hazen, New York, and Rachel F. Brown, Albany,N. Y., assignors to Research Corporation, New York, N. Y., a corporationof New York Application January 7, 1952, Serial No. 265,293 8 Claims.(Cl. 16765) This invention relates generally to antibiotics and theirproduction, and more particularly to a new and useful antibiotic andmethods of producing it.

Most antibiotics known at the present time are characterized by highantibacterial activity with little or no antifungal activity. In aninvestigation of newly isolated actinomycetes for antagonists topathogenic fungi an organism has been found which when cultivated underparticular controlled conditions yields an apparently-intracellularantifungal antibiotic, which is both fungistatic and fungicidal andapparently lacks antibacterial activity.

The organism belongs to a species under the genus Streptomyces, theparticular strain having been isolated from a farm soil in FauquierCounty, Virginia. The species has been designated as Streptomycesnoursei. A pure culture of the organism has been filed with theCollection of Type Cultures of the Division of Laboratories andResearch, New York State Department of Health, Albany, New York, underthe number 48240.

Cultures of the organism have also been filed with the American TypeCulture Collection, wherein they have been assigned the identifyingdesignation: ATCC 11455.

The antibiotic has been designated as nystatin (formerly designatedfungicidin). It is a quite stable, relatively water-insoluble compoundwhich is relatively nontoxic in antibiotically-etfective doses andactive in vivo and in vitro against a wide variety of saprohytic andpathogenic fungi.

Nystatin is obtainable by the following general procedure: the organismStreptomyces noursei is grown in contact with (i. e., in or on) asterile liquid nutrient medium containing a source of assimilable carbonand nitrogen, and the nystatin formed is recovered, preferably from themycelium (i. e. organism growth). -In the preferred method, the myceliumis first separated from the culture liquid; the separated mycelium isextracted with an organic solvent for nystatin, especially a loweraliphatic alcohol; and the solution of nystatin thus obtained is treatedto further purify and concentrate the nystatin therein. Alternatively,the mycelium may be extracted while in the culture liquid, in which casethe organic solvent for nystatin employed must be substantiallywater-insoluble.

In the accompanying drawings Fig. 1 is a flow sheet of a typicalisolation and recovery procedure as described in Example 1; and

Fig. 2 is an infra-red absorption spectrum of the new antibiotic.

The organism Szreptomyces nounsei is characterized by straight, curvedand wavy mycelium, and by round and oval spores produced in chains fromcurved and spiralforminghyphae (loose, closed spirals). When grown onSabourauds glucose agar at 28i1 C. for seven days, the colony is heapedand folded; the aerial mycelium is at first white, then later (atsporulation) gray; and on the under surface, the color is at first lighttan, and

after two weeks it is usually dark brown and the medium n t ds t s PefitO.

2,797,183 Patented June 25, 1957 is darkened throughout. There is nohemolysis on blood agar medium, but the medium is darkened.

The organism Streptomyces noursei is also characterized by thefollowing:

Odor: strong earthy. Gelatin (12%): rapid liquefaction. Sabouraudsglucose agar:

(At 356 C.)good growth, heaped and folded; white aerial mycelium,infrequently becoming rusty pink; in about 50% of the cultures, adiffusible pigment of pomegranate purple color (Ridgeways colorstandards, Plate 12) is produced.

(At 28i1 C.)good growth, heaped and folded; white aerial myceliumbecoming gray; on reverse of growth, pigment is brown and medium isoften darkened throughout. On one occasion in two years a difiusiblepigment, pomegranate purple, was produced in one culture.

(At 40 C.)slight growth, glabrous.

Szapeks agarz' (At 35-6 C.)no growth.

(At 28il C.)growth very scanty; no aerial mycelium; colony small,colorless, flat and deeply embedded.

Asparagin-glucose-agar (synthetic):

(At 356 C.)growth cerebriform in center, with finely-wrinkled periphery;aerial mycelium shellpink and scant; under surface of growth dark gray;small amount of diifusible, shell-pink pigment produced.

(At 28- 1 C.)good growth; gray and white knoblike projections on abackground of more flattened and wrinkled tan growth; on under surfacepigment is dark gray.

Ammonium chloride-glucose-agar (synthetic):

(At 35-6 C.)growth cream-colored; slightly 'heaped and folded in center,and flattened at periphery; no pigment on under surface; no diffusiblepigment.

(At 28:1" C.)growth slightly heavier than at 35-6 C., but otherwise thesame. Hydrolyzed casein-glucose-agar:

(At 356 C.)growth heaped and folded in center, flattened at periphery;aerial mycelium has tannish tint; no ditfusible pigment.

(At 28:1 C.)good growth; dark-gray aerial mycelium; under surface darkgray; no diffusible pigment.

Cellulose agar (:both at 28 21 C. and 35-6 C.): growth very poor.

Starch agar (:both at 28:1 C. and 35-6 C.): good growth of discretecolonies with white aerial mycelium in center and periphery colorlessand embedded; starch hydrolyzed.

Blood agar (both at 28-* -l C. and 35-6 C): good growth; coloniesconvex, lobat-e, with central perforation; heavy, white, chalky aerialmycelium; no hemolysis, but blood is darkened; occasionally glabrouscolony found, which may show pink pigment; on transfer to Sabourau-dsdextrose, such colony may or may not,

3 '(At 28:1 C.)grow-th heaped and folded, with White, chalky, aerialrnycelium; no pigment. Carrot plug:

(At 35-6 C.)growth heaped and folded, chieflyglabrous; small amount ofdirty-white mycelium; no pigment.

(At 28:1 C.)-growth heaped and folded, with white, chalky aerialmycelium.

Nitrites: trace produced from nitrates.

Infusion broth (with 1% glucose, at 28:1 C.): White growth on surface;flocculent sediment; clear broth.

Honey broth: 1

(At 35-6 C.)small amount of white growth on surface; later heavy whitecollar, flocculent sediment.

(At 28:1 C.)heavy white collar; fiocculent sediment; clear broth.

Glucose-tryptone-agar:

(At 356 C.)-growth dry, slightly heaped and folded in center and moreflattened on periphery; aerial mycelium cream-colored; under surfacepinkish; small amount of light-pink diffusible pigment.

(At 28:1 C.)-growth heavier, more heaped and folded than at 35-6 C.;aerial mycelium powdery and gray; under surface tannish-brown; smallamount of tannish-green diffusible pigment.

Glucose-tryptone broth .plus 0.1% agar:

(At 28:1 C.)-surface of medium covered with heavy growth, aerialmycelium heaped, white turning to gray; soluble pigment, pomegranatepurple, rarely produced; HzS produced; slight amount of acid produced.

(At 356 C.)-type of growth same as at 28 C., but less abundant; solublepigment, pomegranate purple, frequently produced.

' The organism Streptomyces noursei may :be grown to produce nystatineither under stationary or surface (aerobic) conditions or undersubmerged aerobic conditions, the latter being effected by means ofagitation and aeration, as well known in the art. The time required formaximum yield of systatin varies with the method of cultivation. In thecase of surface culture, elaboration of nystatin is usually complete inabout 5-7 days; and in the case of submerged culture, in about 3-4 days.The ptimal temperature for production of nystatin is around 28 C., theyield of nystatin dropping off at higher and lower temperatures ofcultivation; and the starting pH may range between about 5.5 and about7.5, but is preferably about 7.0-7.2.

Various liquid nutrient media containing sources of assimilable carbonand nitrogen may be employed. The assimilazble carbon source may be anassimilable polyhydric alcohol (e. g., glycerol) or (preferably) anassimilable saccharide (mono-, di-, or poly-; e. g., glucose). Theassimilable source of nitrogen is preferably an assimilableproteinaceous material, especially a protein degradation or hydrolysisproduct (e. g., tryptone). Other utilizable saccharides include maltoseand lactose; and other utilizable proteinaceous materials include yeastextract, malt extract, casein digest, soybean digest, and corn steepliquor. Preferably, also, the medium includes added essential salts,where necessary, inter alia, potassium, sodium, magnesium and ironsalts.

The concentration and purification ofnystatin may be accomplished in anumber of ways, representative methods being described in detailhereinafter.

The new antibiotic shows no pronounced basic or acidic properties. Noknown salts have been isolated. The antibiotic decomposes when heated inaqueous solutions of acids or alkalis. In methanol solution itwithstands a temperature of 55 C. for 1 hour at pH 5 to 8. It containsthe elements C, H, and N. Chemical analysis shows carbon 58.13%;hydrogen 8.77% and nitrogen 1.76%. All of the nitrogen appears to be inthe amine form. The substance shows no optical rotation. In 0.025%solution in methanol the ultraviolet absorption curve gave 4 t maximumabsorption at 2350 A., 2914 A., 3043 A., and 3193 A. at E=298, 536, 754,and 596 respectively.

The infrared spectrum was obtained on a sample of highly purifiednystatin suspended in hydrocarbon oil using a Perkin Elmer model 12Bspectrometer. The bands obtained are listed below in terms of theirfrequencies (cm- (cm- (CH1."

The antibiotic is almost insoluble in water. It is soluble in methanolto the extent of 0.25 mg./ml., in methanol N/50 hydrochloric acid to theextent of 5 mg./ml., in 70% methanol to the extent of 0.5 mg./m1., in70% ethanol to the extent of 2 mg./ml., and in 60% acetone to the extentof 0.25 mg./ml. In N hydrochloric acid it dissolves to the extent of0.45 mg./ml., and in N sodium hydroxide to the extent of 5 mg./ml. Thesolubility decreases with the purity of the product. It starts todecompose about and does not melt when heated up to 295.

The new antibiotic shows reducing properties in that Benedicts solutionis reduced; bromine water, and iodinepotassium iodide solution aredecolorized by it. The following colors appear with concentrated acids:red violet with sulfuric; pink with phosphoric; yellow withhydrochloric. It gives no biuret or ninhydrin reaction. It produces nocolor with ferric chloride.

IN VITRO ACTIVITY OF NYSTATIN Nature of tests.(All tests performed witha stock suspension of partially-purified nystatin containing 5 mg./rnl.,obtained by dissolving the nystatin in cold N/50 hydrochloric acid andadjusting immediately to pH 7.0- 7.2 with cold 0.1% sodium carbonatesolution, the stock suspension being kept in a refrigerator and used foronly 72 hours.)

(1) Determination of the sensitivity of Cryptococcus neoformans, Candidaalbicans, and other fungi to nystatin: Serial 2-fold dilutions are madein 4.5 ml. glucose-tryptone broth and heated for ten minutes at 70 (forsterilization). To two series of tubes is added, respectively, 0.5 ml.of a saline suspension of C. neoformans and C. albicans inconcentrations of 1,000,000 and 300,000 cells/ml. Readings of visiblegrowth are made after 5, 24, 72 and 96 hours incubation at 28, andsensitivity recorded as the least amount of nystatin inhibiting growth,as evidenced by absence of gross turbidity after 96 hours.

(2) Determination of the antimicrobial activity of nystatin: The agardilution method of Reilly, Schatz and Waksman (J. Bact., 1945, v. 49,585-94) is used, except that infusion agar is employed for the bacterialcultures and the temperature of incubation is 35-6".

(3) Further determination of the effect of nystatin on the growth of C.neoformans: Two sets of serial 2-fold dilutions in glucose-tryptonebroth are inoculated, respectively, with 10 million and 1 millionmicroorganism/ml. After incubation at 3, 4, 5, 24 and 48 hours, aliquotportions are removed and plated on glucose-tryptone agar; and colonycounts are made after 48 hours incubation.

(4) Determination of the efiect of defibrinated horse blood and serum:0.1 ml. of nystatin suspension diluted serially in sterile saline so asto contain from IOU-3.13

micrograms is added to a series of tubes containing 0.25 mil. wholeblood or serum, and to each dilution is added 0.05 ml. of a 48-hourglucose-tryptone broth culture of C. neoformans containing 26 millionmicroorganisms/ml. The tubes are then incubated for 48 hours withfrequent shaking, after which the contents are streaked on plates ofSabourauds glucose agar which are then incubated for 48 hours.

Results of tests.Some results of these tests are given in Tables I andII, hereinafter. They indicate that nystatin is strongly fungistaticagainst a wide variety of saprophytic and pathogenic forms, amounts ofthe order of 1.56-6.25 micrograms/ml. being effective against manyfungi.

TAB LE I [Fungistatic action of nystatm] Sensitivity: micrograms ofnystatin per ml. at 28=l=1 0. Test species 72 hrs. 96 hrs.

'. moformtms 1. 56 1. 56 C. albicans 3.13 3.13

TABLE II [Antiiungal spectrum of nystatim] Least amount inhibiting FungiStrain N 0. growth,

micrograms] Cruptococrus caslella'aii 45232 3. 13 Cruptococcasglati'ais" 4676 1. 56 Candida guillrermondi 45211 3.13 Candida krusei45214 6. 25 C 45213 3. 13 Saccharomz/"es cerem'siae 45217 3.13SporoboZomz/ces salmOnlcolOT 4550 3. 13 Schizosaccharomyces octosporus"45231 1. 56 Endomucopsis fibuliger 45230 3. 13 Geotricham lactic 474626. 25 Aspergillus fumigatus 50248 6. 25 Pmirillium mime/nu 43281 3. 13.Penicillium 3p 50526 13 Penicillium clamforme 49470 3.13 RMZOPUSnigrtcans 50524 3. 13 Fasarium 8P. 50527 3. 13 Alternaria 813-- 50523 1.56 Cephalosporium sp- 45226 25 Phoma sp 50522 6. 25 C'eratostomellaul'mi (plant pathogen) 50525 6. 25 Hormodendru'm sp 4893 3. 13Histoplasma rapsulalum (yeastlike) 4894 1. 56 Blastomzlces dermatitidis(yeastlikeL. 45223 1. 56 Paracoccirioides brasiliensis (yeastlike45224 1. 56 Coccidioldes immitis (spherules) 50521 6. 25 Cryptacoccusneoformam 45215 1. 56 C albic 4657 3. 13 Trichophyton mentagrophytes45141 6.25 flrichophylrm rubru'm 4516 6. 25 Trichophz ton r0saceam 49743.13 Epidermophylon floccosu'm 44253 1. 56 Micrasporam audouini 4896 3.13 lldi rasporum canis 4817 13 Sporotrz'chum schenckii (mycellal) like)50251 13 Monosporium apz'ospermum 45221 100 Allescheria boy/[iii 48102100 Phialophoru aerrucosa 45229 13 Inhibition of growth of the followingbacteria is not obtained even with 100 micrograms/ml. of the partiallypurified nystatin: Staphylococcus aureus (No. 45142), Streptococcushemolyticus (No. 44131), Bacillus subtilis (No.'45137), Bacillus cereusvar. mycoia'es (No. 48294), Salmonella typhosa (No. 38351), Shigellaparadysenteriae (No. 45300), Bacterium mucosum capsulatum (No. 4767),and Bacillus circulans (No. 48205). Growth of a strain of Mycobacteriumtuberculosis (No. 50539) freshly isolated from a human lung is notinhibited in Dubos medium containing 100 micrograms mystatin/ml.

-Nystatin is strongly fungicidaltas well as fungistatic). In thepresence of one million microorganisms (C. neoformans), 6.25microgram/ml. mystatin brings about a progressive decline in thepopulation and no viable cells are detected at 24 hours; and with 12.5micrograms/mL,

no viable cells are detected at 3 hours. With a tenfold increase inmicroorganisms, and 12.5 micrograms/ml. nystatin, no viable cells aredetected at 4 hours.

- Incubation of nystatin in the presence of defibrinated horse blood orserum gives no indication of diminishing or otherwise modifying itsactivity.

IN VIVO ACTIVITY OF NYSTATIN Nature of zests.-All tests were performedwith stock suspension described hereinbefore, diluted with sterilephysiologic salt solution, and in white mice (Albany strain) of 20-25 g.which were infected by intraperitoneal and'intravenous injections ofsaline suspensions 'of C. neoformans or intraperitoneal injections ofHistoplasma capsulatum in large doses.

Results of tests-(I. T0xicity).-The approximate LDSO of thepartially-purified nystatin administered intraperitoneally is between 20and 26 mg./kg.; injected subcutaneously, however, 2 g./kg. does notkill, but there is induration and necrosis at the injection site. Micereceiving 473 mg./kg. show no evidence of necrosis. Mice receiving atotal of 735 mg./kg. over a period of four weeks show no ill effects,and compare favorably with an untreated control group in appearance andweight (the nystatin being administered subcutaneously orintraperitoneally in doses of 200-400 micrograms, five days each in thefirst and second weeks, four days the third week, and three days thefourth week).

(II. Therapeutic activity).It was definitely established that miceinjected with large amounts of C. neoformans or H. capsulatum and givenrepeated parenteral does of nystatin have a milder form of infection,and that life is prolonged beyond that of the controls.

The following examples are illustrative of the invention (alltemperatures being in centigrade, all solutions not otherwise identifiedbeing aqueous, and all strain numbers given being those of theaforementionedv collection of type cultures):

. Example 1 (a) A glucose-tryptone medium is prepared with dispurity,free of other sugars, starch, proteins, alcohol and heavy metals), 0.5%tryptone, 0.1% agar, 0.2% K2HPO4, 0.2% NaCl, and 0.001% FeSO4, and beingadjusted to pH 7.0-7.2 by addition of normal hydrochloric acid solution;and 400 ml. aliquots of the medium are placed in 3-liter Erlenmeyer orFernbach flasks. The flasks are then plugged with cotton, sterilized byautoclaving at 15 lbs. pressure (121") for 20 minutes, cooled,inoculated with a Streptomyces noursei inoculum (7-10 day old pellicleproduced on the same medium), and incubated at 28:1 for five to sevendays.

(b) The pellicle is harvested from 5- to 7-day-old cultur'es, drained onwire strainers, and macerated in a Waring blendor for 1 minute withmethanol, 1 ml./ g. The material is centrifuged at 10 C. and the extractremoved. Two more extractions are made by stirring the residue for 1hour with methanol in the same proportion. The extracts are chilled at012 C. for at least 24 hours. They are then clarified by centrifugationat below 10 C. and the solvent removed in vacuo at below 45 C. Acetonemay be substituted for methanol using pure solvent for the firstextraction and 50% for subsequent extractions. The residue is suspendedin 50 ml. each of butanol and 0.85 sodium chloride solution per l-2liters of extract and chilled at 0:2 C. for at least 24 hours. Thesuspension is separated by centrifugation at below 10 C. and filtrationinto 3 parts. The bottom aqueous phase and the top butanol phase arediscarded. The insoluble material at the interface contains the majorpart of the nystatin. It is washed on the filter with 0.85% salinesaturated with butanol to remove inert pigments, and next with 0.85saline to remove butanol and more pigments. The washed residue isdissolved in a minimum of methanol using heat (temperature up to 55 C.)if

equal volume of 0.85 sodium chloride solution.- After 2 hours at i2 C.the precipitate (A) is collected by centrifugation at below C. and thesupernate treated further with 0.85 sodium chloride solution up to atotal of 2 volumes. After standing at least 24 hours at 0i2 C. theprecipitate (B) is collected by centrifugation at below 10 C. Each ofthe two saline precipitates, designated A and B, respectively, isseparately dissolved in methanol (with warming up to 55 C. if desirable)and reprecipitated with one and two volumes of 0.85% sodium chloridesolution, respectively. After chilling to 012 C. the precipitates arecollected by centrifugation at below 10 C. Each of the two salineprecipitates is separately dissolved in methanol (with warming up to 55C. if desirable), and reprecipitated with 4 volumes of ethyl acetate.After standing overnight at 0i2 C. the precipitates are collected anddried with ether. The dry products represent purified antibiotic. Bothproducts are yellow; that obtained by one volume saline precipitation ismore highly colored. The antibiotic may be recovered in one step by asingle precipitation with two-three volumes of saline.

Example 2 (a) A medium is prepared, inoculated and incubated asdescribed in Example 1 (a). a

(b) The surface growth in each flask is then harvested,- heated at 70-72for ten minutes, and pressed between filter papers to remove adheringmedium, the pressed mycelium containing about 70-83% moisture (asdetermined by loss on heating at about 105 for five hours). Methanol isthen added to the pressed mycelium, in the proportion of 1.5-3 ml./g.,the mixture is stirred in a Waring blender for a half minute at roomtemperature,-

centrifuged cold (below about 10), and the supernatant decanted. Theresidue is extracted with the same amount of methanol for 2-3 hours atroom temperature; and the residue'obtained on centrifuging this mixtureand decanting the supernatant is again extracted in the same manner. Thepooled methanol extracts (supernatant's) are chilled to 0-5,centrifuged, and the resulting methanol solution of nystatin isrecovered by decantation.

(c) Ethyl acetate is added to the methanol solution of nystatin, firstin a proportion of 0.3-2.0 times the volume of the methanol toprecipitate inert material (removed by filtration or centrifugation),then to a total of 4-5 times'the volume of the methanol, and theresulting precipitate is recovered. The precipitate is then washed with0.85%NaCl solution, dissolved in methanol, and the ethyl-acetateprecipitation repeated. The precipitate thus obtained is again dissolvedin methanol, and the solution mixed with 3-4 volumes ethyl ether; andthe resulting precipitate is separated, washed with ether, dried andground to a powder.

The product thus obtained'in a yield of about 30-50 mg/liter. broth (2.partially-purified nystatin) is a fine yellow powder that can be storedin the cold for several months without loss of activity. It is slightlysoluble in methanol, 65-70% ethanol, wet butanol, 70% acetone, propyleneglycol, ethylene glycol monomethyl ether, and water at pH 2-3 or 8-9;less soluble in 95% ethanol, butanol, and acetone; and insoluble inether, chloroform, ethyl acetate, amyl acetate, benzene, petroleum ether(low boiling), and water (around neutrality). It contains about 1.5%Kjeldahl nitrogen (at that stage of purification), evidences no sulfuror halogen content, shows strong reducing properties, fails to react intests for protein, and gives no color with ferric chloride; and itsmolecule is relatively small, being'ultrafiltrable through fine gradocolmembranes (thin membranes of collodion or similar substances graded asto porosity). Aqueous solutions of the productprepared at pH 2 and'pH 9are very unstable. The product may be used as atherapeutic '8 agentwithout further purification, or-rnay be further treated to concentrateand/orpurify the ny statin-therein.

Example 3 as described in Example 1(a).

(b) The whole culture is then mechanically stirred for 2-3 hours withA-l volume of n-butanol at room temperature, and the n-butanol phaseseparated and centrifuged cold (it necessary), The aqueous phase ismechanically stirred for 2-3 hours with /8- /2 volume n-butanol, then-butanol phase separated, and the remaining aqueous phase againextracted in the same manner with n-butanol. The pooled n-butanolextracts are chilled to 0-5", centrifuged, and the supernate decanted.The

n-butanol is then removed from the supernate in vacuo at below 45, theresidue extracted with methanol, and the methanol extract is furthertreated as described for the methanol solution of nystatin in section 0of Example 2. The partially-purified nystatin thus obtained isqualitatively the same as that obtained in Example 2.

Example 4 and it is adjusted to pH 7.0-7.2 by addition of normalsodiumhydroxide solution, and sterilized by autoclaving at 15 lbs. pressure(121) for 20 minutes. The nystatin preparation thus obtained isqualitatively the same as that obtained in Example 1 and describedhereinbefore, but is obtained in lower yield.

Example 5 (a) A glucose-tryptone medium is prepared as described inExample 1(a) (except that the agar is omitted). ml. aliquots of themedium are placed in 300 m1. Erlenmeyer flasks, and the flasks areplugged with cotton, sterilized by autoclaving at 15 lbs. pressure (121)for 20 minutes. The flasks are then cooled, inoculated with a salinesuspension of a 7-9 day old culture of Streptomyces noursei onSabourauds dextrose medium, and incubated at 28i1 for 3-4 days on ashaking machine which gives the flasks a rotary and tilting motionsimulating that given by hand shaking.

(b) The whole culture is then treated as described in Example 1, thepartially-purified nystatin thus obtained The following additionalmedia, inter alia, may be employed for the submerged-culture productionof nystatin as described in Example 5:

A. Medium described in Example 5 with agar omitted.

B. Medium described in Example 1 with agar omitted and with crudedextrose in place of the highly-pure dextrose. i

C. Medium described in Example 1 with agar omitted and with crudemaltose in place of thedextro's'e. I

D. Medium described in Example 1 with agar omitted and with malt extractin place of the dextrose.

E. Medium described in Example 1"with agar omitted and with proteosepeptone in place of the trypton'e.

F. Distilled water containing following ingredients in percentagesindicated: 7 V

9. G. Distilled water containing following ingredients in percentagesindicated:

The following other variations may be made in the procedure described inExample 2 without qualitatively atfecting the production and recovery ofnystatin:

1. Heating of the surface growth after harvesting may be omitted.

2. Amyl acetate may be used in place of ethyl acetate.

3. Extraction may be efiected by grinding the surface growth with themethanol, or percolating methanol through the surface growth.

4. n-butanol, ethanol, or a mixture of equal parts of methanol andethanol may be substituted for the methanol.

The production of nystatin may, of course, be efiected on a largerscale, utilizing where applicable the wellknown techniques and apparatusemployed in the commercial, large-scale production of other antibiotics,both by stationary or submerged culture.

The invention may be variously otherwise embodied within the scope ofthe appended claims.

This application is a continuation-in-part of our application Serial No.208,978 filed February 1, 1951, now abandoned.

We claim:

1. The method of producing a fungistatic substance from the organismStreptomyces noursei grown in contact with a sterile liquid nutrientmedium containing sources of assimilable carbon and nitrogen, whichcomprises separating the mycelium of the organism from the culturemedium, extracting the separated mycelium with a solvent of the groupconsisting of lower alcohols and lower alkyl ketones, and recoveringfrom the extract the fungistatic substance extracted thereby from themycelium.

2. The method as defined in claim 1 wherein the fungistatic substance isrecovered from the extract by evaporating the solvent.

3. The method as defined in claim 1 wherein the fungistatic substance isprecipitated from the extract by the addition of an aqueous solution ofsodium chloride.

4. The method as defined in claim 1 wherein the fungist-atic substanceis precipitated from the extract by the addition of a lower alkylacetate.

5. The mehod as defined in claim 1 wherein the fungistatic substancerecovered from the extract is purified by repeated solution in a loweralcohol and precipitation from the solution with an aqueous solution ofsodium chloride.

6. The method as defined in claim 1 wherein the fungistatic substancerecovered from the extract is purified by 10 suspending it in a mixtureof butanol and an aqueous solution of sodium chloride, and thereafterseparating the insoluble material including the fungistatic substance atthe interface between the butanol phase and the aqueous phase of themixture.

7. The method as defined in claim 1 wherein the mycelium is extractedwith methanol, the methanol extract is evaporated leaving a solidresidue, the solid residue is treated with a mixture of butanol and anaqueous solution of sodium chloride, the solid material is separatedfrom the interface between the butanol and the aqueous phases of themixture and is thereafter subjected to repeated solution in methanol andprecipitation with an aqueous solution of sodium chloride.

8. An antifungal substance obtained from the mycelium of a culture ofStreptomyces noursei, which is relatively insoluble in water, sparinglysoluble in the lower alcohols and lower alkyl ketones, contains carbon,hydrogen and nitrogen, gives no color with ferric chloride, fails toreact in tests for protein, is active in vivo and in vitro against manysaprophytic and pathogenic fungi and exhibits characteristic absorptionbands in the infrared region of the spectrum when suspended in solidform in a hydrocarbon oil at the following frequencies expressed inreciprocal centimeters: 3356, 2898, 1706, 1623, 1575, 1543, 1466, 1387,1349, 1292, 1267, 1178, 1130, 1107, 1073, 1031, 1005, 962, 947, 932,907, 848, 801, 772, 752, 740, and 710.

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Arnstein et al.: Iour. of Gen. Microbiology, No. 3, May 1948, pp.111-122 (particularly pp. 112, 117 and 122).

Hazen et al.: Science, Oct. 13, 1950, p. 423.

Science Newsletter, Oct. 21, 1950, vol. 58, No. 17, p. 262.

Newcomer et al.: Jour. of Invest. Dermatology, May 1954, pp. 431-440.

Hazen et al.: Proc. Soc. Exptl. Biol. & Mei, vol. 76, No. 1, Jan. 1951,pp. 93-97.

Raubitscheck et al.: Antibiotics & Chemotherapy, vol. 2, No. 4, pp.179-183.

Brown et al.: Antifungal Substances from Actinomycetes Annual Report,Div. of Laboratories and Research, New York State Dept. of Health,19-21, 1949.

Articles on Nyst-atin in Antibiotics Annual 1953- 1954, published byMed. Encyclopedia Inc., 1953, pp.

' 191-214 (pp. 199-209 pertinent).

8. AN ANTIFUNGAL SUBSTANCE OBTAINED FROM THE MYCELIUM OF A CULTURE OFSTREPTOMYCES NOURSEI, WHICH IS RELATIVELY INSOLUBLE IN WATER, SPARINGLYSOLUBLE IN THE LOWER ALCOHOLS AND LOWER ALKYL KETONES, CONTAINS CARBON,HYDROGEN AND NITROGEN, GIVES NO COLOR WITH FERRIC CHLORIDE, FAILS TOREACT IN TESTS FOR PROTEIN,M IS ACTIVE IN VIVO AND IN VITRO AGAINST MANYSAPROPHYTIC AND PATHOGENIC FUNGI AND EXHABITS CHARACTERISTIC ABSORPTIONBANDS IN THE INFRARED REGION OF THE SPECTRUM WHEN SUPSPENDED IN SOLIDFORM IN A HYDROCARBON OIL AT THE FOLLOWING FREQUENCIES EXPRESSED INRECIPROCAL CENTIMETERS: 3356, 2898, 1706, 1623, 1575, 1543, 1466, 1387,1349, 1292, 1178, 1130, 1107, 1073, 1031, 1005, 962, 947, 932, 907, 848,801, 772, 752, 740, AND 710.